CN113215143A

CN113215143A - Extraction method of total RNA of micro-tissue of ascidian larva

Info

Publication number: CN113215143A
Application number: CN202110459240.8A
Authority: CN
Inventors: 李世国; 战爱斌; 程佳威; 李茜
Original assignee: Research Center for Eco Environmental Sciences of CAS
Current assignee: Research Center for Eco Environmental Sciences of CAS
Priority date: 2021-04-27
Filing date: 2021-04-27
Publication date: 2021-08-06

Abstract

The invention discloses a method for extracting total RNA of a micro-tissue sample of ascidian larvae, which relates to the field of molecular biology research and mainly comprises the following steps: separating a micro tissue sample by a tungsten needle; ultrasonic crushing and cracking; ethanol precipitation of total RNA; washing total RNA with ethanol and Tris buffer; RNase-free water elutes total RNA. The quality inspection of the extracted total RNA is carried out by utilizing real-time fluorescent quantitative PCR and full transcriptome amplification technology, which shows that the method can efficiently extract the total RNA in the micro sample of the sea squirt larva, the quality of the obtained RNA is higher, and the requirements of related molecular biological experiments are met. The invention can solve the problem of extracting total RNA of micro tissue or organ samples at the larval stage of organisms, and provides technical support for molecular biology research such as marine organism transcriptome or genome and the like.

Description

Extraction method of total RNA of micro-tissue of ascidian larva

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a method for extracting total RNA from a micro tissue sample of ascidian larvae, aiming at successfully extracting the total RNA from the micro tissue sample at the juvenile stage of marine organisms for subsequent molecular biology experiments such as transcriptome or genome.

Background

Ascidians (Ascidians) are tunicates belonging to the phylum chordata and the subphylum urospora, and are widely distributed in various types in the global sea area. The life history of ascidians can be divided into two developmental stages, juvenile and adult. The larva of ascidian hatched from embryo is tadpole-shaped, and has a ridge cable at its tail and papilla structure at its head. After the sea squirt larva freely swims for hours, the sea squirt larva is attached to artificial facilities such as underwater rocks and ship bodies by utilizing the mastoid structure, and after retrograde metamorphosis, the spinal cord degenerates and disappears, and the sea squirt larva grows to an adult stage and permanently adheres to filter food life. The ascidians are important biological groups in the transition from invertebrates to vertebrates in the biological evolution history, have special classification positions, become model organisms in the research fields of genetics, development, evolutionary biology and the like, and have high research value. Meanwhile, most species of ascidians have the characteristics of biological invasion and biological fouling, and have great threats to the global marine ecosystem. The sea squirt can successfully invade the sea area all over the world by means of the transportation of ships and the like across the sea area, and the health and the stability of the sea ecosystem of the invaded area are threatened. The adhered ascidians often cause the problem of biological fouling, and cause serious economic loss to national economic important industries such as ship transportation, aquaculture, water conservancy facilities and the like. Therefore, the research on the biology of ascidians, especially the research on the larval stage of free swimming, is not only a focus of attention in the biological field, but also an important way to solve the problems of marine ecological environment such as biological invasion and biofouling, and the related research results are widely representative.

The analysis of gene expression characteristics of certain specific tissues or organs by high-throughput technical means such as genomics and transcriptomics is now a feasible method for studying physiological and biochemical processes and molecular regulation mechanisms of marine organisms. The application of this technology requires the extraction of high quality RNA from specific tissues or organs. The conventional total RNA extraction methods for ascidian organisms comprise a Trizol method, various kit extraction methods and the like, wherein the kit extraction methods can be divided into a magnetic bead method, an adsorption column method and the like according to a nucleic acid adsorption mode, and are successfully used for extracting total RNA of ascidian larva. However, these existing extraction methods can only use the whole individual of ascidian larva, often tens or even thousands of individuals mixed together as a sample for RNA extraction. The gene expression has tissue specificity, the acquisition of the gene expression characteristics of a single tissue or organ is the key point for deeply analyzing the gene function and the regulation mechanism of ascidians, and a specific extraction method for the total RNA of the tissues or organs at the juvenile stage of the ascidians is not available at present. The sea squirt in the larval stage is tiny, the total length is only about 1mm, the total number of cells of the whole individual is about 2600, a single tissue or organ is only dozens or hundreds of cells, the sample size is small, and the obtaining of a complete tissue or organ sample is difficult. In addition, the external side of the ascidian larva has protective structures such as a capsule, and the like, so that cell lysate in the conventional total RNA extraction kit cannot enter the larva to directly play a role, and the extraction efficiency is often low. Due to the low concentration, RNA obtained from micro-tissue samples cannot be subjected to quality detection by conventional methods such as gel electrophoresis and spectrophotometer. These problems make it difficult to obtain total RNA from these micro-tissues or organs by conventional extraction methods. Therefore, there is a need to develop an efficient method for extracting total RNA from micro-tissue or organ samples of ascidians at the larval stage.

Obtaining total RNA of high quality ascidian micro-tissue samples is a critical need for research in a plurality of disciplines, such as research on the origin, evolution and development patterns of tissue and organs from the developmental biology perspective, research on the invasion and fouling problems of marine organisms from the ecological perspective, and the like. In view of the mode biological attributes of the ascidians and the universality of the molecular biology research method, the establishment of the extraction method of the total RNA of the micro-tissue sample at the juvenile stage of the ascidians has better reference significance for the establishment and improvement of the extraction method of the total RNA of the micro-tissue of other biological species.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide a method for effectively extracting total RNA from a trace amount of ascidian juvenile tissue or organ samples. The extraction method has high efficiency and simple operation, obtains high quality RNA, can meet the requirements of subsequent molecular biology experiments, and is suitable for extracting total RNA from trace biological samples.

The extraction method of total RNA of micro-tissues of ascidian larvae provided by the invention comprises the following steps:

1) adding ascidian larva into sterilized seawater dripped on a glass slide in advance, dissecting and separating a target tissue or organ sample, and transferring the separated tissue or organ sample to cell lysis solution;

2) placing cell lysate containing tissue or organ samples on ice, and carrying out ultrasonic crushing to obtain homogenate;

3) standing the homogenate liquid at room temperature for 5-10min, centrifuging at 20000g for 3min, discarding the precipitate, and transferring the supernatant to a centrifuge tube;

4) adding 70% (volume content) ethanol into the supernatant, mixing, transferring to RNA adsorption column, centrifuging for 30s at 9000g, and discarding the centrifugate;

5) adding 70% (volume content) ethanol into the adsorption column, washing, centrifuging for 30s at 9000g, and discarding the centrifugate;

6) adding washing buffer solution into the adsorption column, centrifuging for 30s at 9000g, and discarding the centrifugate;

7) adding 80% (volume content) ethanol into the adsorption column, centrifuging for 2min at 9000g, and discarding the centrifugate;

8) opening the adsorption column at 20000g, centrifuging for 5min, and discarding centrifugate;

9) putting the adsorption column into a new centrifuge tube, adding RNase-free water into the center of the adsorption column, standing, centrifuging at 20000g for 1min, and collecting the eluted liquid to obtain total sample RNA.

In the step 1) of the method, the ascidian larvae are fresh ascidian larvae;

the sterilized seawater is prepared by the following method: filtering natural seawater with 0.45 μm filter membrane, and sterilizing at 121 deg.C for 30 min.

The dissection is carried out under a dissecting mirror, and a tungsten needle with the diameter of 5 mu m is used for dissection;

the isolated tissue or organ is viscera, head, tail, papilla, etc.;

the components of the cell lysate are as follows: 4mol/L guanidinium isothiocyanate, 25mmol/L sodium citrate (pH7.0), 0.5% sodium dodecyl sarcosinate, 40mmol/L Dithiothreitol (DTT);

the volume of the cell lysate may be 300-400 μ L;

in the step 2), the ultrasonic disruption is carried out by an ultrasonic cell disruptor;

the amplitude transformer of the ultrasonic cell disruptor is phi II type.

The parameters of the ultrasonic crushing are set as follows: the total crushing time is 2min, the working time r is 1.0s, the pause time P is 3.0s, and the power is 25-27%;

in the step 3), the volume of the centrifugal tube can be 1.5 mL;

in step 4), the volume of the 70% (volume content) ethanol can be 300-400 μ L (equal volume to the cell lysate);

the mixing is realized by blowing and beating through a pipettor;

in the step 5), the volume of 70% ethanol for washing can be 300-;

in step 6), the washing buffer is: 50mM Tris (pH 6.8) and absolute ethanol in a ratio of 1: 4 (volume ratio);

the volume of the washing buffer can be 400-600 mu L;

in the step 7), the volume of the 80% ethanol can be 400-;

in the step 9), the new centrifugal tube is a new 1.5mL centrifugal tube;

the volume of RNase-free water may be 12-15. mu.L;

the standing time can be 1-2 min.

The cell lysate used in the above method also belongs to the protection scope of the present invention.

The components of the cell lysate are as follows: 4mol/L guanidinium isothiocyanate, 25mmol/L sodium citrate (pH7.0), 0.5% sodium dodecylsarcosinate, 40mmol/L Dithiothreitol (DTT).

Based on the technical scheme, the invention provides a technical method for micro sample RNA of ascidian larvae, which comprises the steps of directly carrying out ultrasonic disruption on dissected micro tissues or organs in cell lysate, centrifuging to remove insoluble substances, adding the insoluble substances into an RNA adsorption column, washing to remove impurities, and finally eluting to obtain a pure RNA sample. The verification proves that the method of the invention has relatively simple operation, small RNA loss and good quality of the obtained RNA, and can be used for subsequent molecular biological experiments and analysis work.

Drawings

FIG. 1 is a graph showing the results of real-time fluorescent quantitative PCR amplification analysis in example 2, wherein four genes are respectively 18S rRNA gene and 3 genes related to the adhesion function of ascidians.

FIG. 2 is a graph of the results of the amplification of the whole transcriptome in example 2 by mass spectrometry and electrophoresis.

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.

The invention provides a method for extracting total RNA of micro-tissues of ascidian larvae, which comprises the following steps:

1) adding sea squirt larva (5 sea squirt larva) into sterilized seawater dripped on a glass slide in advance, dissecting and separating a target tissue or organ sample, and transferring the separated tissue or organ sample to a cell lysate;

the washing buffer is: 50mM Tris (pH 6.8) and absolute ethanol in a ratio of 1: 4, preparing a mixed solution.

The invention provides a technical method for micro sample RNA of ascidian larva, which comprises the steps of directly carrying out ultrasonic disruption on dissected micro tissues or organs in cell lysate, adding into an RNA adsorption column after removing insoluble substances by centrifugation, removing impurities by washing, and finally eluting to obtain a pure RNA sample. The verification proves that the method of the invention has relatively simple operation, small RNA loss and good quality of the obtained RNA, and can be used for subsequent molecular biological experiments and analysis work.

Example 1 extraction of Total RNA from Trace sample of ascidians

In the process of realizing the invention, the inventor firstly carries out microdissection by using a superfine tungsten needle under a dissecting mirror aiming at the obtained micro sample of the sea squirt larva to obtain a specific tissue or organ; transferring the dissected trace sample into cell lysate, directly performing physical disruption on the sample mixed solution by using an ultrasonic cell disruption instrument, and centrifuging to remove insoluble substances; transferring the supernatant mixed with ethanol to an RNA adsorption column; washing with ethanol and Tris buffer solution in sequence; finally, the mixture is eluted by using a proper amount of RNase-free water.

The specific operation steps are as follows:

A. the experiment uses young ascidian hyalophia hyalopecurosa (Cionarobusta) to obtain the artificial insemination antibody after culturing for 24h at 18 ℃.

B. Filtering the collected natural seawater with 0.45 μm filter membrane, and sterilizing with high pressure steam sterilizing pot at 121 deg.C for 30min to obtain sterilized seawater.

C. 5 fresh sea squirt larvae are added into sterilized seawater which is dripped on a glass slide in advance, a target tissue sample (papilla structure) is quickly dissected and separated by a tungsten needle with the tip diameter of 5 mu m under a dissecting mirror, and the separated tissue sample is immediately transferred into 350 mu L of cell lysate by using a pipette, wherein the cell lysate comprises the following components: 4mol/L guanidinium isothiocyanate, 25mmol/L sodium citrate (pH7.0), 0.5% sodium dodecylsarcosinate, 40mmol/L Dithiothreitol (DTT), wherein DTT is added at the time of use.

D. The cell lysate containing the tissue sample is placed on ice and physically disrupted with an ultrasonic cell disruptor to obtain a homogenate. Wherein, select phi II type amplitude transformer, the parameter setting: the total crushing time is 2min, the working time r is 1.0s, the intermittent time P is 3.0s, and the power is set to be 25-27%. The whole crushing process was operated on ice.

E. The crushed homogenate was left at room temperature for 5min, centrifuged at 20000g for 3min, the precipitate was discarded, and the supernatant was aspirated and transferred to a new 1.5mL centrifuge tube.

F. Add 350. mu.L 70% (volume content) ethanol to the supernatant, pipette the mixture, transfer the whole mixture to an RNA adsorption column, centrifuge for 30s at 9000g, and discard the centrifugate.

G. 350 μ L of 70% (volume content) ethanol was added to the adsorption column, and the column was washed, centrifuged at 9000g for 30 seconds, and the centrifuged solution was discarded.

H. mu.L of washing buffer was added to the adsorption column, and 9000g was centrifuged for 30s, and the centrifuged solution was discarded. Wherein, the washing buffer solution comprises the following components in percentage by weight of 1: 4 (by volume) of 50mM Tris (pH 6.8) and absolute ethanol.

I. mu.L of 80% (volume content) ethanol was added to the adsorption column, 9000g was centrifuged for 2min, and the centrifugate was discarded.

J. The column was uncapped and centrifuged at 20000g for 5min, and the centrifugate was discarded.

K. Putting the adsorption column into a new 1.5mL centrifuge tube, adding 15 μ L RNase-free water to the center of the adsorption column, standing for 2min, centrifuging at 20000g for 1min, and collecting the eluted liquid to obtain total sample RNA.

Example 2 detection of Total RNA in Trace amount of ascidian lacteus samples

The method detects the total RNA of the extracted ascidian papilla micro-sample, and comprises real-time fluorescence quantitative PCR and full transcriptome amplification analysis so as to verify the quality and the availability of the total RNA obtained by the extraction method provided by the invention.

(1) Real-time fluorescent quantitative PCR detection

Reverse transcription was performed using the total RNA of ascidian papilla obtained in example 1 as a template to obtain cDNA, and real-time fluorescent quantitative PCR analysis was performed. The method comprises the following specific steps:

A. reverse transcription was performed using the Prime Script II 1st Strand cDNA Synthesis Kit reverse transcription Kit from TaKaRa. Reaction system 1 was placed in a 0.2mL centrifuge tube. System 1 had a total volume of 10 μ L, including: mu.L dNTP, 1. mu.L oligodT, 8. mu.L total RNA eluate.

B. The system 1 was treated at 65 ℃ for 5 min. After the reaction was completed, the product was quickly taken out and stored on ice.

C. Reaction system 2 was placed in another 0.2mL centrifuge tube. System 2 had a total volume of 10 μ L, including: 4 μ L of 5 Xprime Script II buffer, 0.5 μ L of RNase Inhibitor, 0.5 μ L of Prime Script II RTase, 5 μ L of ddH₂O。

D. Adding the system 2 into the reacted system 1, blowing, beating and mixing uniformly, treating at 42 ℃ for 45min, and treating at 95 ℃ for 5 min. Obtaining the cDNA product. The reaction product was stored at 4 ℃.

E. The cDNA obtained above is used for real-time fluorescent quantitative PCR detection. The fluorescent quantitative PCR is carried out by adopting a SYBR Green I fluorescent labeling method in a FastStart Essential DNA Green Master kit of Roche company, and the amplification system is 20 mu L, which comprises 10 mu L of visual 1, 8 mu L of visual 2, 0.5 mu L of forward and reverse primers and 1 mu L of cDNA template.

F. The fluorescent quantitative PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 600 s; denaturation at 95 ℃ for 10s, denaturation at 57 ℃ for 15s, and extension at 72 ℃ for 15s, for a total of 45 cycles; the final 95 ℃ treatment for 10s, 65 ℃ treatment for 60s, and 97 ℃ treatment for 1s were used to generate melting curves.

G. The real-time fluorescent quantitative PCR analysis selects 1 18S rRNA gene of ascidians hyalopecuroides as reference gene and 3 genes related to ascidian adhesion function as target genes. Each gene was set up with 3 technical repeats. The corresponding primer sequences of these 4 genes are as follows:

18S rRNA

forward primer sequence 5'-TCTGCCCTATCAACTTTCGTC-3'

Reverse primer sequence 5'-GATGTGGTAGCCGTTTCTCAG-3'

Seq1

Forward primer sequence 5'-CTCAATGCCAATGTAACCAGG-3'

Reverse primer sequence 5'-CAACGCCGATAGTGACCAAAC-3'

Seq2

Forward primer sequence 5'-GCGATAACCGTCATAGCGTAG-3'

Reverse primer sequence 5'-TGCGTTGAAGCAGGAGAAGC-3'

Seq3

Forward primer sequence 5'-GCCGTGCTTTCAACTCACTC-3'

Reverse primer sequence 5'-CGGTGCAAGATTTGGTACGC-3'

The results of the real-time fluorescent quantitative PCR analysis are shown in FIG. 1. The amplification tendency of the internal reference gene and the 3 adhesion function related genes Seq1, Seq2 and Seq3 is obvious, the expression difference of each gene is obvious, the difference between the technical repetition of each gene is small, the amplification result is stable, and the result shows that the total RNA quality of the ascidian mastoid process extracted by the method is good, and the requirement of real-time fluorescence quantitative PCR analysis can be met.

(2) Whole transcriptome amplification assay

Whole transcriptome amplification analysis was performed using the total RNA of ascidian papilla obtained in example 1 as a template. The method comprises the following specific steps of using a Smart-seq2 method:

A. and adding a primer. The total reaction system is 4.3 mu L, comprising 0.3 mu L of total RNA, 2 mu L of buffer solution, 1 mu L of oligo-dT primer and 1 mu L of dNTP mix, the mixture is mixed evenly and reacted for 3min at 72 ℃, and the product is placed on ice;

B. and (5) reverse transcription. The reaction was carried out using the PrimeScript II 1st Strand cDNA Synthesis Kit of TaKaRa. The total reaction system was 10. mu.L, and included 4.3. mu.L of the first-step reaction product, 0.5. mu.L of SuperScript II reverse transcriptase, 0.25. mu.L of RNAse inhibitor, 2. mu.L of SuperScript II first-strand buffer, 0.5. mu.L of DTT, 2. mu.L of Betaine, 0.06. mu.L of MgCl₂0.1. mu.L of TSO, 0.29. mu.L of nucleic-free water; the reaction procedure is as follows: treating at 42 deg.C for 90min, at 50 deg.C for 2min, and at 42 deg.C for 2min, setting the above reactions for 10 cycles, and treating at 70 deg.C for 15min to obtain the final productThe reverse transcription product of (4 ℃ C.) was stored.

And C, PCR amplification. The total reaction system was 25. mu.L, containing 10. mu.L of reverse transcription product, 15. mu.L of PCR mix (12.5. mu.L of KAPA HiFiHotStartStreacyMix, 0.25. mu.L of IS PCR primers, 2.25. mu.L of nucleic-free water); the reaction procedure is as follows: pre-denaturation at 98 ℃ for 3min, denaturation at 98 ℃ for 20s, annealing at 67 ℃ for 15s, and extension at 72 ℃ for 6min, wherein the above reactions are set for 18 cycles, and finally treatment at 72 ℃ for 5min, and the obtained PCR product is stored at 4 ℃.

The obtained PCR product was subjected to quality detection using a high throughput sequencing library quality control Kit (DNF-474-33-HS NGS Kit, Fragment 1-6000bp), and the analysis results are shown in FIG. 2. The result shows that the amplified product fragments are mainly distributed between 0.7 kb and 3.0kb, the main peak of the product is distributed between 1.0 kb and 2.0kb, the size of the highest peak is 1434bp, and the amplification quality of the whole transcriptome is better. The detection result shows that the total RNA quality of the micro sample of ascidian mastoid extracted by the method is good, and the micro sample can be used for library construction such as omic sequencing and the like.

The existing RNA extraction method aims at the whole ascidian individual. There is no general and efficient method for RNA extraction from individual organs or tissues of ascidians with a smaller sample size. Because of the presence of capsule structures in vitro in ascidian larvae and the relatively small number of organ or tissue samples, the commonly used RNA extraction steps/methods are not efficient. The invention provides a definite RNA extraction method for ascidian larva tissues or organs for the first time.

The foregoing is only a preferred embodiment of this invention and it should be noted that numerous changes and modifications could be made herein by one of ordinary skill in the art without departing from the general principles of the invention and, it is intended that such changes and modifications be considered as within the scope of the invention.

Claims

1. A method for extracting total RNA of micro-tissues of ascidian larvae comprises the following steps:

4) adding 70% ethanol into the supernatant, mixing, transferring to RNA adsorption column, centrifuging at 9000g for 30s, and discarding the centrifugate;

5) adding 70% ethanol into the adsorption column, washing, centrifuging at 9000g for 30s, and discarding the centrifugate;

7) adding 80% ethanol into the adsorption column, centrifuging for 2min at 9000g, and discarding centrifugate;

2. The method of claim 1, wherein: in the step 1), the sterilized seawater is prepared by the following method: filtering natural seawater with 0.45 μm filter membrane, and sterilizing at 121 deg.C for 30 min.

3. The method according to claim 1 or 2, characterized in that: in step 1), the dissection is performed under a dissecting scope, and is performed by using a tungsten needle with the tip diameter of 5 μm.

4. The method according to any one of claims 1-3, wherein: the isolated tissue or organ is an internal organ, head, tail or papilla.

5. The method according to any one of claims 1-4, wherein: the components of the cell lysate are as follows: 4mol/L guanidinium isothiocyanate, 25mmol/L sodium citrate, pH7.0, 0.5% sodium dodecyl sarcosinate, 40mmol/L dithiothreitol;

the volume of the cell lysate was 300-400. mu.L.

6. The method according to any one of claims 1-5, wherein: in the step 2), the ultrasonic disruption is carried out by an ultrasonic cell disruptor;

the amplitude transformer of the ultrasonic cell disruptor is phi II type;

the parameters of the ultrasonic crushing are set as follows: the total crushing time is 2min, the working time r is 1.0s, the pause time P is 3.0s, and the power is 25-27%.

7. The method according to any one of claims 1-6, wherein: in the step 4), the volume of the 70% ethanol is 300-;

in the step 5), the volume of 70% ethanol for washing is 300-;

in step 6), the washing buffer is: 50mM Tris (pH 6.8) and absolute ethanol in a ratio of 1: 4, preparing a mixed solution according to the proportion;

the volume of the washing buffer was 400-.

8. The method according to any one of claims 1-7, wherein: in the step 7), the volume of the 80% ethanol is 400-;

in the step 9), the volume of the RNase-free water is 12-15 μ L;

the standing time is 1-2 min.

9. A cell lysate comprising the following components: 4mol/L guanidinium isothiocyanate, 25mmol/L sodium citrate, pH7.0, 0.5% sodium dodecyl sarcosinate, 40mmol/L dithiothreitol.

10. Use of a cell lysate according to claim 9 for the extraction of total RNA from ascidian larvae microtissue.